Telomeres are stabilizing elements located at the ends of the chromosomes. Telomere maintenance is central to unlimited replication - a key hallmark of the uncontrolled growth of cancer. Telomeres shorten with each cell division; a process that causes eventual cell cycle exit in normal cells. In contrast, many cancers preserve their telomeres by co-opting the telomere-specific reverse transcriptase telomerase, which has been intensively studied as a potential target for anti-cancer therapy. However, significant numbers of cancers lack telomerase; relying instead on a homologous recombination-based telomere maintenance mechanism termed ALT (Alternative Lengthening of Telomeres). In a recent broad survey of over 90 cancer subtypes, the ALT mechanism was found to be utilized in 46% of the subtypes; with notable prevalence in sarcomas, pancreatic neuroendocrine tumors and certain tumors of the central nervous system. Importantly, the ALT phenotype was found to be completely cancer-specific, thus it represents an attractive anti-cancer target. Unfortunately, relatively little is currently known concerning potentially exploitable molecular underpinnings of the ALT mechanism. Recently, we discovered nearly perfect correlations between the ALT phenotype and recurrent cancer-associated somatic mutations in the genes ATRX and DAXX in a variety of cancers. Together, the protein products of these genes form a chromatin remodeling complex that functions as a histone chaperone, targeting incorporation of the histone variant H3.3 into G-rich repetitive regions of the genome, including the telomeres which are normally maintained in a repressed heterochromatic state. Our primary hypothesis is that defects in the ATRX/DAXX complex compromise the formation and/or maintenance of heterochromatin at the telomeres, thus rendering them permissive for homologous recombination which is integral to the ALT telomere maintenance mechanism. The overall objectives of this proposal are to elucidate the roles of defective ATRX and DAXX in the ALT phenotype, with an eye towards exploiting these defects for potential therapeutic purposes. These objectives will be accomplished through three specific aims. In aim 1, we will use in vitro cell- based functional assays to test the hypothesis that loss of ATRX or DAXX is sufficient for the induction of ALT via a reduced heterochromatic state at the telomeres. In aim 2, we will use transgenic mouse models to test the hypothesis that loss of ATRX or DAXX fosters malignant transformation in the pancreas, and that the tumors arising will engage the ALT pathway. In aim 3, we will test the hypothesis that an unbiased, in vitro cellular toxicity screen of an established chemical compound library will reveal synthetic lethality specific to ALT-positive cells, or cells in which ATRX or DAXX has been abrogated. Successful completion of these aims will provide insights into the roles of ATRX and DAXX mutations in ALT and tumorgenesis; thus paving the way for the development of new effective treatment strategies to help eradicate this significant subset of cancers.